The goal of this project is to use genetic approaches to determine the function of the protein recently found to be mutated in patients with the Wiskott-Aldrich Syndrome (WAS), a severe X-linked disease that is characterized by a B-and T-cell immunodeficiency, eczema, bloody diarrhea, and thrombocytopenia. Two strategies will be used to develop a mouse model of the WAS. First they have targeted and disrupted the murine homologue of the human WAS gene in murine embryonic stem (ES) cells to generate WAS-deficient stem cells. For a rapid analysis of the effects of the WAS-deficiency on lymphoid development, they have generated somatic chimeras in the recombinase activating gene-2-deficient (RAG2-/-) complementation system. RAG2-deficient mice are unable to rearrange immunoglobulin and T-cell receptor genes and therefore lack mature B and T cells. WAS-deficient ES cells have been introduced into RAG2-/- blastocyts; all of the B- and T-cells from resulting chimeric mice are of ES cell origin; thus one can analyze the role of WASp selectively in lymphocyte development and function. Preliminary studies suggest that murine WAS- lymphocytes have defects that closely resemble the human disease. WAS-deficient ES cells will also be used to generate germline WAS- mice for the analysis of more widespread effects and for long term studies. Experiments will be carried out to determine whether the germline WAS-deficient mice and the WAS-deficient RAG2-deficient chimeras are valid models to study the human disease. In vivo complementation analyses will be performed with various regions of the WAS gene to define and characterize functional domains of the protein. This application is for a Mentored Clinical Scientist Development Award to an Applicant who has received graduate training in microbiology and immunology, and has completed post graduate training in internal medicine and clinical gastroenterology. The applicant's long term goals are to establish and direct his own independent basic research program in studies related to diseases that affect the human immune system with clinical interests in diseases that affect the mucosal immune system. Accordingly, these studies are co-sponsored by Dr. Frederick Alt from the Department of Genetics at the Howard Hughes Medical Institute and the Center for Blood Research and by Dr. Daniel Podolsky from the Gastrointestinal Unit at the Massachusetts General Hospital, both at Harvard Medical School. (End of Abstract)